The role of de novo ceramide synthesis in the mechanism of action of the tricyclic xanthate D609

The cytotoxic effects of several chemotherapeutic drugs have been linked to elevated de novo ceramide biosynthesis. However, the relationship between the intracellular site(s) of ceramide accumulation and cytotoxicity is poorly understood. Here we examined the relationship between the site of ceramide deposition and inhibition of protein translation and induction of apoptosis by the antitumor/antiviral xanthate, D609. In Chinese hamster ovary (CHO)-K1, HEK-293, and NIH-3T3 cells, D609 caused rapid (1–5 min) and sustained eukaryotic initiation factor 2α (eIF2α) phosphorylation followed by apoptosis after 24 h. Concurrently, D609 stimulated de novo ceramide synthesis and increased ceramide mass 2-fold by 2 h in CHO-K1 cells. In D609-treated CHO-K1 cells, sphingomyelin synthesis was stimulated by brefeldin A, and C5-DMB-ceramide transport to the Golgi apparatus was blocked, indicating ceramide accumulation in the endoplasmic reticulum (ER). However, D609-mediated eIF2α phosphorylation, inhibition of protein synthesis, and apoptosis in CHO-K1 cells were not attenuated by fumonisin B1 or l-cycloserine. Interestingly, short-chain ceramide promoted eIF2α phosphorylation and inhibited protein synthesis in CHO-K1 cells, indicating that the effectiveness of endogenous ceramide could be limited by access to signaling pathways.Thus, expansion of the ER ceramide pool by D609 was not implicated in early (eIF2α phosphorylation) or late (apoptotic) cytotoxic events. The cytotoxic effects of several chemotherapeutic drugs have been linked to elevated de novo ceramide biosynthesis. However, the relationship between the intracellular site(s) of ceramide accumulation and cytotoxicity is poorly understood. Here we examined the relationship between the site of ceramide deposition and inhibition of protein translation and induction of apoptosis by the antitumor/antiviral xanthate, D609. In Chinese hamster ovary (CHO)-K1, HEK-293, and NIH-3T3 cells, D609 caused rapid (1–5 min) and sustained eukaryotic initiation factor 2α (eIF2α) phosphorylation followed by apoptosis after 24 h. Concurrently, D609 stimulated de novo ceramide synthesis and increased ceramide mass 2-fold by 2 h in CHO-K1 cells. In D609-treated CHO-K1 cells, sphingomyelin synthesis was stimulated by brefeldin A, and C5-DMB-ceramide transport to the Golgi apparatus was blocked, indicating ceramide accumulation in the endoplasmic reticulum (ER). However, D609-mediated eIF2α phosphorylation, inhibition of protein synthesis, and apoptosis in CHO-K1 cells were not attenuated by fumonisin B1 or l-cycloserine. Interestingly, short-chain ceramide promoted eIF2α phosphorylation and inhibited protein synthesis in CHO-K1 cells, indicating that the effectiveness of endogenous ceramide could be limited by access to signaling pathways. Thus, expansion of the ER ceramide pool by D609 was not implicated in early (eIF2α phosphorylation) or late (apoptotic) cytotoxic events. Ceramide is recognized as a key mediator of cell stress and apoptosis. Numerous studies have demonstrated that generation of ceramide, as either an early or a late response to various stimuli, is involved in subsequent downstream events that control cell survival [reviewed in refs. (1Hannun Y.A. Functions of ceramide in coordinating cellular responses to stress.Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1495) Google Scholar, 2Hannun Y.A. Luberto C. Ceramide in the eukaryotic stress response.Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar)]. An emerging theme suggests that ceramide acts as an index or coordinator of cellular stress responses whereby elevation of ceramide beyond a certain threshold regulates protein phosphatases (3Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid.J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 4Ruvolo P.P. Gao F. Blalock W.L. Deng X. May W.S. Ceramide regulates protein synthesis by a novel mechanism involving the cellular PKR activator RAX.J. Biol. Chem. 2001; 276: 11754-11758Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) or kinases (5Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis.Nature. 1996; 380: 75-79Crossref PubMed Scopus (1714) Google Scholar, 6Lee J.Y. Hannun Y.A. Obeid L.M. Ceramide inactivates cellular protein kinase Calpha.J. Biol. Chem. 1996; 271: 13169-13174Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7Westwick J.K. Bielawska A.E. Dbaibo G. Hannun Y.A. Brenner D.A. Ceramide activates the stress-activated protein kinases.J. Biol. Chem. 1995; 270: 22689-22692Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar) involved in mitogenesis, apoptosis, or senescence. By virtue of its central position in sphingolipid metabolism, ceramide is regulated by multiple synthetic and catabolic processes. Initially, ceramide generation in response to cytokine-activated neutral sphingomyelinases (SMases) (8Dobrowsky R.T. Jenkins G.M. Hannun Y.A. Neurotrophins induce sphingomyelin hydrolysis. Modulation by co- expression of p75NTR with Trk receptors.J. Biol. Chem. 1995; 270: 22135-22142Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 9Kim M.Y. Linardic C. Obeid L. Hannun Y. Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation.J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar, 10Kolesnick R.N. Haimovitz-Friedman A. Fuks Z. The sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor, Fas, and ionizing radiation.Biochem. Cell Biol. 1994; 72: 471-474Crossref PubMed Scopus (141) Google Scholar) was identified as an important feature of signaling cascades. More recently, genotoxic drugs and environmental stress have been shown to increase ceramide by de novo biosynthesis (11Perry D.K. Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). A variety of apoptotic and cytotoxic agents, such as etoposide (11Perry D.K. Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), SDZ PSC 833 (12Cabot M.C. Han T.Y. Giuliano A.E. The multidrug resistance modulator SDZ PSC 833 is a potent activator of cellular ceramide formation.FEBS Lett. 1998; 431: 185-188Crossref PubMed Scopus (55) Google Scholar), daunorubicin (13Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar), phorbol ester (14Garzotto M. White-Jones M. Jiang Y. Ehleiter D. Liao W.C. Haimovitz-Friedman A. Fuks Z. 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Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). B1 or inhibited and in cell with that daunorubicin elevated de novo ceramide synthesis, that ceramide was not involved in the of factor by activates and expression in and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, induction of cell stress and apoptosis by chemotherapeutic of the pathway and elevation of endogenous ceramide downstream signaling A that is the intracellular of the ceramide that is in response to The for the of ceramide synthesis to the of the endoplasmic reticulum K. J. The of is the of the endoplasmic reticulum in J. PubMed Scopus Google Scholar). Ceramide is to the Golgi apparatus by an is to sphingomyelin by synthase M. M. K. for endoplasmic apparatus of ceramide for sphingomyelin synthesis in Chinese hamster ovary Cell Biol. 1999; PubMed Scopus Google Scholar). In induction of ceramide synthesis is not by in or synthesis (12Cabot M.C. Han T.Y. Giuliano A.E. The multidrug resistance modulator SDZ PSC 833 is a potent activator of cellular ceramide formation.FEBS Lett. 1998; 431: 185-188Crossref PubMed Scopus (55) Google Scholar, J. S. L. L.M. of de novo ceramide biosynthesis in tumor necrosis cell Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). suggests that ceramide synthesis either its to by subsequent in the pathway and in the or is to sphingolipid not important for as that generation of ceramide in various by expression of effects apoptosis S. Hannun Y.A. Obeid L.M. of a pool of sphingomyelin J. 2001; PubMed Scopus Google Scholar). D609 was shown to that could be a to ceramide and subsequent effects signaling C. G.M. Hannun Y.A. effects of sphingomyelin and the of in and Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). D609 was identified as an and of E. The D609 is a of 1996; Google Scholar). to the of the to a of not been identified or shown to be inhibited by D609. D609 been in cell to the effects of drugs or to K. C. E. G. of signal transduction by an and Cell PubMed Scopus Google Scholar, L. of D609 in the of a key role for Lett. 2001; PubMed Scopus Google Scholar, G. B. necrosis factor alpha activates the phosphorylation of and kinase in of 2001; PubMed Scopus Google Scholar), in have the of D609 been In was recognized that D. D.A. D609 ionizing by as a potent 2001; Google Scholar, J. K. of by is by PubMed Scopus Google Scholar) and A. J. A. and of D609 PubMed Scopus Google Scholar). a was that was to D609 M.J. A. M. A novel of PubMed Scopus Google Scholar), that the of D609 could be to its have that D609 could have involved in signaling C. G.M. Hannun Y.A. effects of sphingomyelin and the of in and Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. in 2000; PubMed Scopus Google Scholar, S. May C. D. The transport of the of its with Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). D609 have of action inhibition that for its Here we that D609 ceramide synthesis and a rapid phosphorylation of eukaryotic initiation factor 2α (eIF2α) followed by apoptosis after 24 h. However, elevated endogenous ceramide synthesis and ceramide mass accumulation in the ER was not involved in eIF2α phosphorylation, inhibition of protein synthesis, or apoptosis. that the effects of D609 could be linked to stress-induced phosphorylation of eIF2α and inhibition of protein synthesis, effects sphingolipid a response to cell that the intracellular site of ceramide accumulation is an important of its The of and were was B1 and were A and kinase and were was and were was and were were Chinese hamster ovary HEK-293, and NIH-3T3 cells were in in and cells were in and NIH-3T3 cells were in were in in of and were h of were in the D609 or was in and to the was in and to for a of fumonisin B1 was in and to a of or and was in and to the of D609 de novo sphingolipid and synthesis, cells were for h in A to with in the with and various to for and cells were with and in and was after of with sphingomyelin synthesis in Chinese hamster ovary 1995; Full Text PDF PubMed Google Scholar). was by of in a of ceramide, and were to in for h were with and by in a of of short-chain ceramide and in Chinese hamster ovary 1995; PubMed Scopus Google Scholar). and were identified by with an and by and ceramide mass were by the kinase J. R. of in and and Biol. Chem. Full Text PDF PubMed Google Scholar) and to cell protein with the Biol. Chem. Full Text PDF PubMed Google Scholar). D609 or cells were with in the and by for The cell were in and by a and was by of the for The was in A to a of synthase and were as of palmitoyltransferase in Chinese hamster ovary PubMed Scopus Google Scholar, of to sphingolipid PubMed Scopus Google Scholar). The ceramide C5-DMB-ceramide was to ceramide transport the ER to the Golgi apparatus by a S. M. M. M. S. K. A novel of ceramide the endoplasmic reticulum to the site of sphingomyelin Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in A A was to cells and were h of for cells were with C5-DMB-ceramide with in for were with followed by with for The was by cells with and the cells in for cells were with with and in and Cell were a with an and and eIF2α were by with protein was with a A. Golgi and phosphorylation of protein in and 2001; Full Text Full Text PDF PubMed Google Scholar). and were by a cells were in 2 and and a by for of to were with for h in and and were with in and for h were with or to and were by the of eIF2α in inhibition of protein translation initiation its of the factor phosphorylation in PubMed Scopus Google Scholar). of eIF2α be by kinases in response to a variety of stress and in 1999; Full Text Full Text PDF PubMed Scopus Google Scholar)]. D609 was to have and we the of could be mediated phosphorylation of eIF2α and subsequent of protein studies that D609 effects eIF2α phosphorylation and sphingolipid and as was in cell HEK-293, and were with D609 for to 2 and the phosphorylation of eIF2α was a of was after D609 to cells, and the cell a between and The of eIF2α phosphorylation in response to an of ER and a activator of eIF2α phosphorylation M. May W.S. a cellular activator for protein kinase during stress Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), after 2 h was to D609 h. of eIF2α phosphorylation by D609 was by a in protein synthesis that was sustained for 2 h D609 the phosphorylation of in to we examined phosphorylation of and in in an increase in mass by and is regulated by the and sphingolipid of the cell A. Golgi and phosphorylation of protein in and 2001; Full Text Full Text PDF PubMed Google Scholar, effects of sphingomyelin and transport protein phosphorylation and Golgi Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and by stress and D609 increased phosphorylation of to as by a to the mass K. of phosphorylation of the protein by brefeldin 1998; PubMed Scopus Google Scholar). for 2 h not an early D609 is phosphorylation of eIF2α and inhibition of protein A that eIF2α phosphorylation was stimulated by short-chain ceramide phosphorylation of PKR activator an activator of the protein kinase P.P. Gao F. Blalock W.L. Deng X. May W.S. Ceramide regulates protein synthesis by a novel mechanism involving the cellular PKR activator RAX.J. Biol. Chem. 2001; 276: 11754-11758Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). ceramides stress kinases (5Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis.Nature. 1996; 380: 75-79Crossref PubMed Scopus (1714) Google Scholar, 7Westwick J.K. Bielawska A.E. Dbaibo G. Hannun Y.A. Brenner D.A. Ceramide activates the stress-activated protein kinases.J. Biol. Chem. 1995; 270: 22689-22692Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, S. M. M. M. S. K. A novel of ceramide the endoplasmic reticulum to the site of sphingomyelin Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), was that pathway could be for of phosphorylation of and inhibition of with that various chemotherapeutic drugs activate ceramide synthesis, that D609 could eIF2α phosphorylation by endogenous ceramide studies in CHO-K1 cells that D609 caused a in after not with inhibition of synthase involved in a pathway for C. G.M. Hannun Y.A. effects of sphingomyelin and the of in and Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the effects of D609 de novo sphingolipid synthesis have not been the that of ceramide synthesis or inhibition of synthase could to the in sphingolipid in response to ceramide and sphingolipid synthesis was in CHO-K1 cells with D609 for to 2 h by with D609 caused a rapid of ceramide that a of by h. Interestingly, D609 increased by to D609 caused a inhibition of synthesis not The of D609 and ceramide to be a response in cells, a increase in and ceramide was in to a NIH-3T3 cells increased de novo ceramide synthesis in increased ceramide CHO-K1 cells were with D609 for to 2 h and ceramide and mass in was by the kinase D609 caused a increased in ceramide mass by h and a increase to 2-fold by 2 h. The increase in ceramide mass in response to D609 was by fumonisin an of ceramide that was de novo D609 to a of by The in mass was not by fumonisin of D609 effects sphingolipid in ceramide biosynthesis were by CHO-K1 cells were with D609 or D609 fumonisin and ceramide and was fumonisin B1 inhibited ceramide synthesis and elevated with inhibition of ceramide of cells with fumonisin B1 the increase in ceramide caused by D609. the in ceramide synthesis D609 and fumonisin B1 was by a increase in that to ceramide synthase were stimulated by D609. The of the in sphingolipid synthesis, is stimulated by genotoxic drugs by (11Perry D.K. Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). D609 a CHO-K1 cells with D609 were for in CHO-K1 cells with D609 was increased by between and was the to control by h. of D609 to CHO-K1 cell not a of the not The in of synthase in D609-treated CHO-K1 cells was the 2 h and was inhibited by h the of Luberto C. G.M. Hannun Y.A. effects of sphingomyelin and the of in and Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) that D609 inhibited synthase by to the in not the in and that D609 sphingolipid synthesis and ceramide mass accumulation increased The of endogenous ceramide is transport to in the cell various signaling or be is ceramide accumulation of de novo synthesis is in the ER or to ceramide accumulation in D609-treated cells was to the could be to the ER and Golgi and ceramide to synthase A. A. E. increase of sphingomyelin for the action of the in Biol. Chem. Full Text PDF PubMed Google Scholar). with in CHO-K1 cells A Chinese hamster ovary cells to by Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Chinese hamster ovary cells the protein synthesis of sphingomyelin in response to 1999; Full Text Full Text PDF PubMed Google Scholar), increased synthesis ceramide D609 caused a elevation in synthesis of ceramide, and to a of and of cells with and D609 in a in accumulation with a in synthesis, that of the ceramide in the ER was to the Interestingly, of and D609 increased by However, the increase in was with the in ceramide and of D609 or promoted a increase in synthesis, the of the was not The accumulation of ceramide in the ER of D609-treated cells and the to that ceramide the ER is inhibited or between the ceramide C5-DMB-ceramide was as a of ceramide transport to the Golgi apparatus R. C. M. of to and in 2000; PubMed Google Scholar). CHO-K1 cells were with or D609 for 2 h to with C5-DMB-ceramide for transport of the to the Golgi apparatus was after for In control cells, C5-DMB-ceramide the ER and in to the Golgi apparatus after In D609-treated cells, C5-DMB-ceramide was in a and was of after that transport to the Golgi apparatus was to inhibition of C5-DMB-ceramide was the of in a ER pool of endogenous ceramide in D609-treated cells, CHO-K1 cells were with fumonisin B1 to endogenous ceramide However, the C5-DMB-ceramide in cells was to that of D609-treated cells and D609 and fumonisin B1 a the of the Golgi as by for was to that of C5-DMB-ceramide in control cells not to the in or cells either or that D609 endogenous ceramide in the ER by of de novo synthesis, we was linked to the phosphorylation of eIF2α shown in In eIF2α phosphorylation was in D609-treated CHO-K1 cells that were with fumonisin B1 or the in to the increase in ceramide and D609 increased phosphorylation of eIF2α after However, fumonisin B1 D609 of eIF2α CHO-K1 cells a increase in eIF2α phosphorylation to not the indicating that signaling that to ceramide were in cells. in phosphorylation of eIF2α shown in were with protein translation as by or not the inhibition of protein translation by and not the inhibited The was not eIF2α phosphorylation inhibited protein synthesis of ceramide by increased de novo synthesis or is with or for apoptosis. was that ceramide in response to not protein could apoptosis. of of the that D609 for 24 h caused limited induction of apoptosis in the cell with a 2 h with the protein kinase of cells with fumonisin B1 or not in D609-treated CHO-K1 or cells. In cells, the of by D609 was inhibited by fumonisin B1 not by l-cycloserine. of in and cells was by the Thus, and apoptosis in response to D609 is not de novo ceramide have the relationship between of ceramide synthesis in the ER and its subsequent transport to the Golgi and phosphorylation of eIF2α and inhibition of protein translation by the D609. that D609 stimulated the sphingolipid to elevated ceramide was not for of eIF2α phosphorylation and apoptosis. ceramide mass accumulation in the of D609 was to the and D609 a stress response that in rapid inhibition of translation and induction of apoptosis. of protein synthesis and induction of apoptosis could be to the and of D609. D609 was identified as a of inhibition of the as a of not been or shown to be inhibited by by in cellular and that inhibition by D609 of an synthase involved in a pathway could ceramide and C. G.M. Hannun Y.A. effects of sphingomyelin and the of in and Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, C. Hannun Y.A. a of intracellular of ceramide and during sphingomyelin synthase for the Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the effects of D609 de novo sphingolipid synthesis that the caused rapid ceramide, by a increase in de novo ceramide synthesis was stimulated by the not the synthase involved in de novo is the that D609 stimulated synthesis the increase in ceramide, and synthesis was stimulated by D609 in the of synthase in D609-treated cells was either not or inhibited late is to the inhibition of synthase by D609 in C. G.M. Hannun Y.A. effects of sphingomyelin and the of in and Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). However, we that D609 inhibited synthase in CHO-K1 cell in and in CHO-K1 cells, D609 caused a inhibition of of by not A for is that synthase involved in and de novo synthetic or de novo ceramide generation in response to D609 with the of of to that D609 stimulated de novo ceramide synthesis the with the ceramide synthase fumonisin B1 elevated ceramide synthesis and mass in response to D609. the of fumonisin B1 to D609-treated cells in a in indicating that a to ceramide synthase was in of D609-treated cells a increase in to that for apoptotic (11Perry D.K. Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, A. M.C. J. S. M. Ceramide signaling in cell apoptosis.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that D609 activates the and in sphingolipid synthesis, to a increase in sphingolipid synthesis and elevated ceramide the mechanism of is the of in response to suggests a mechanism involving a apoptotic or stress-induced The between ceramide that of and that ceramide the ER was inhibited by D609. is by with synthesis in D609-treated cells by the ER and Golgi apparatus and ceramide with was in ceramide by The to either that synthase was with that not ER ceramide was to the or that ceramide been the ER and was not by of the that of de novo ceramide synthesis by D609 in a in the Ceramide accumulation in the ER could have either inhibition of or of the pathway M. M. K. for endoplasmic apparatus of ceramide for sphingomyelin synthesis in Chinese hamster ovary Cell Biol. 1999; PubMed Scopus Google Scholar). endogenous ceramide, transport of the ceramide C5-DMB-ceramide the ER to the Golgi was inhibited in the of D609 and not by ceramide with fumonisin B1 the that D609 transport of ceramide the ER or a to the Golgi D609 have effects and protein been to transport to the in CHO-K1 cells S. May C. D. The transport of the of its with Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with stimulated phosphorylation of eIF2α by an stress-activated kinase P.P. Gao F. Blalock W.L. Deng X. May W.S. Ceramide regulates protein synthesis by a novel mechanism involving the cellular PKR activator RAX.J. Biol. Chem. 2001; 276: 11754-11758Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The mechanism phosphorylation of its to and of the kinase of several eIF2α kinases M. May W.S. a cellular activator for protein kinase during stress Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, a cellular activator of protein Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). to with short-chain ceramide in CHO-K1 cells stimulated phosphorylation of eIF2α and inhibited protein translation endogenous generation of ceramides in response to D609 was not linked to eIF2α is the of of fumonisin B1 and D609-mediated eIF2α phosphorylation and inhibition of protein and a between ceramide accumulation min) and eIF2α phosphorylation (1–5 In we that with D609 apoptosis, as by of the D609 to induce apoptosis, ceramide synthesis with fumonisin B1 or not between in effects with endogenous ceramide could be by access to signaling pathways. short-chain ceramide cells and access cellular and signaling pathways. In that endogenous ceramides the de novo pathway be in the ER and not involved in subsequent signaling events. of of ceramide is by a ceramide generation by expression of in S. Hannun Y.A. Obeid L.M. of a pool of sphingomyelin J. 2001; PubMed Scopus Google Scholar). In that ER expression of in cells increased ceramide not induce apoptosis. The of the that the site of generation and of endogenous ceramide important its A between the effects of ceramide or ceramide the by and ceramide in the In the of of ceramide a pool of ER ceramide that access to of the cell and is not involved in stress or apoptotic with was by and The and for during the of was by a of and and a and of brefeldin A Chinese hamster ovary eukaryotic initiation factor 2α endoplasmic reticulum protein protein kinase PKR activator sphingomyelin palmitoyltransferase

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